Synthesis of useful compounds in plants, e.g., therapeutic agents, chemical feedstocks, cosmetics and other consumer goods, and genetic engineering for crop improvement, for example, genetically engineered pathogen-resistant plants, are two areas of plant biotechnology that are of increasing economic importance and research interest around the globe (Miele, 1997; Franken et al., 1997; Beachy, 1997; Ma et al., 1996; Estruch et al., 1997; Palmgren, 1997). In addition, the availability of plant genomic and EST libraries is changing the potential for commercial plant biotechnology (Briggs et al., 1997; McKusick, 1997; and Evans. et al., 1997). In particular, because many plant traits, such as yield, harvest index, heterosis, and environmental stress tolerance, are regulated by the concerted action of several genes, access to gene sequence tags and genomic libraries provides a means to identify, or a ready source for, genes that are of commercial interest.
Vectors based on several plant viruses, for example, tobacco mosaic virus (TMV), Cowpea Mosaic Virus (CpMV), Bromoviruses (e.g., BMV) and Potyviruses (Yusibov et al., 1997; Reimann-Phillip et al., 1993; Porta et al., 1996; Johnson et al., 1997; Kollar et al., 1993; Pruss et al., 1997), are being developed to introduce selected genes into plants for the synthesis of useful compounds. Virus-based vectors offer several distinctive advantages for gene transfer that complement Agrobacterium-mediated transformation and particle bombardment methods. Viral vectors are easily manipulated; they can be transferred by simple inoculation and they permit high levels of expression in transfected plants. However, the existing plant virus-based vector systems have their limitations. They can cause disease in their plant hosts, ranging from minor to catastrophic. Also, they naturally spread in nature, from one plant to another in the field, and in some cases they can be spread through pollen or seed to the next generation. Moreover, many of these viruses have narrow host ranges and have limited utility due to variable expression of foreign genes.
Nodaviruses are small, non-enveloped icosahedral viruses of insect origin. Mosquitoes (Culex tritaeniorhynchus, Aedesa egypti, Culex tarsalis, Aedes albopictus and Toxorhynchites amboinesis), larvae of the Indian meal moth and the black carpet beetle (Plodia interpunctella: Lepidoptera and Attagenus piceus: Coleoptera), larvae of the wax moth (Galleria mellonella: Lepidoptera) and honey bee (Apis mellifera) are some of the insect hosts known to be infected by nodaviruses and in which virus infection causes paralysis and death (Tesh, 1980; Bailey and Scott, 1972). Exemplary nodaviruses include Black beetle virus (BBV), Flock House virus (FHV), Boolarra virus (BOV) and Nodamura virus (NOV). These viruses are the only known messenger sense, bipartite genome RNA viruses of higher or lower animals (for a review, see Hendry, 1991). Bipartite genome plant viruses are known, but their genome parts are encapsidated in separate virions (Bruening, 1977).
The natural hosts of Nodaviruses are insects, however, Nodaviruses can multiply in animals (for example, NOV can infect mice and possibly pigs, see Bailey and Scott, 1972; Scherer and Hurlbut, 1967; Scherer et al., 1968), and recently have been found in marine fish (Mori et al., 1992; Nishizawa et al., 1995; Frerichs et al., 1996), although FHV does not multiply at all in higher animal cells. In addition, FHV readily multiplies in plants without causing disease symptoms (Selling et al., 1990). FHV can be introduced to plants by mrechanically rubbing the leaves or may be introduced by spraying the leaves with a solution containing the virus. However, FHV moves poorly or not at all from the site of infection.
Thus, what is needed is a vector to systemically or locally transmit a desired or preselected nucleic acid segment or sequence to plants without causing disease to those plants.
The present invention provides a recombinant viral nucleic acid-based gene transfer vector useful to transmit genes to plants and animals, e.g., insects. Preferably, the vector is derived from a virus having a bipartite genome which comprises single strand linear RNA, e.g., Nodavirus (Hendry, 1991), Dianthovirus, Tobravirus (Matthews, 1991) and the like. Thus, the invention provides a biologically active gene transfer vector, comprising linked nucleic acid sequences. The linked nucleic acid sequences include a nucleic acid sequence derived from the 5xe2x80x2 end of a viral nucleic acid, e.g., the 5xe2x80x2 end of Nodavirus RNA-1 or Nodavirus RNA-2; a nucleic acid sequence comprising at least one nucleic acid segment of interest; and a nucleic acid sequence derived from the 3xe2x80x2 end of a viral nucleic acid, e.g., the 3xe2x80x2 end of Nodavirus RNA-1 or Nodavirus RNA-2. The nucleic acid sequence comprising the nucleic acid segment of interest preferably encodes a plant virus movement protein, a plant virus coat protein (e.g., a native coat protein or a modified coat protein having different properties than the native coat protein), a growth hormone, a toxin, e.g., a sublethal toxin such as an auxin or a Photorhabdus toxin, a cytokine, disease resistance, pest resistance, male sterility, antigenic sites on the surface of a virus useful for vaccine production, or pesticide resistance, and/or comprising a synthetic gene, targeted antisense RNA sequences/enzymes to modify plant properties, a marker gene or a selectable marker, or any combination thereof. Preferably, a nucleic acid segment comprises sequences modified for enhanced expression in plants. The nucleic acid segment(s) are preferably linked so as to result in a fusion polypeptide. Alternatively, nucleic acid sequences encoding proteolytic sites may be introduced between each nucleic acid segment. Optionally, each nucleic acid segment may be operably linked to a transcription control unit, e.g., a promoter. A vector of the invention may be combined with other animal virus-based vectors, plant virus-based vectors or other insect virus-based vectors, for example, a tobacco mosaic virus-based vector (Beachy, 1997), a baculovirus-based vector (Schneemann et al., 1993) or a vaccinia virus-based vector (Ball, 1992).
A preferred vector of the invention comprises Nodavirus nucleic acid at the 5xe2x80x2 and 3xe2x80x2 ends of the vector and encodes a plant virus movement protein (MP). MP assists in the translocation of a virus or viral nucleoprotein (genetic material plus xe2x80x9ccarrier proteinxe2x80x9d) from plant cell to plant cell. Thus, the expression of a MP in a plant cell having a vector of the invention leads to the transmission of vector sequences to surrounding plant cells, which results in a local or transient transmission and expression of those sequences. Thus, where transient or localized expression is desirable and sufficient (as in high throughput screening/genomics), the transmission of a vector of the invention can dramatically reduce the time to screen for expression compared to a transgenic approach. In addition, since Nodaviruses, Dianthoviruses and Tobraviruses can be transmitted by simple mechanical rubbing of leaves, vectors based on these viruses may be used on existing varieties of plants without the need to prepare transgenic versions. In particular, the invention provides an approach to crop improvement which avoids the time-consuming and expensive methods needed to prepare transgenic crops and avoids the potential for contamination of seed banks and germplasm collections with foreign genes, which is associated with transgenic approaches.
The invention further provides a method to introduce a preselected nucleic acid sequence to a host cell. The method comprises contacting a host cell with a vector of the invention in an amount effective to express the preselected nucleic acid sequence without producing infectious virus particles. If the host is an organism, the absence of infectious virus completely eliminates the threat for any unexpected and undesirable consequences for the host, e.g., insects or human beings. Preferably, for plant hosts, the contact includes mechanical rubbing, e.g., of the leaves.
The invention also provides compositions useful to transfer genes to host cells. One embodiment of a composition of the invention includes (a) an amount of nucleic acid encoding a viral polymerase (replicase), such as Nodavirus RNA-1; and (b) an amount of recombinant nucleic acid. The recombinant nucleic acid comprises a nucleic acid sequence derived from the 5xe2x80x2 end of a viral nucleic acid, e.g., the 5xe2x80x2 end of Nodavirus RNA-2; nucleic acid sequence comprising at least one nucleic acid segment of interest; and a nucleic acid sequence derived from the 3xe2x80x2 end of a viral nucleic acid, e.g., the 3xe2x80x2 end of Nodavirus RNA-2. The presence of nucleic acid encoding a viral replicase, e.g., RNA-1 of Nodavirus, in a composition of the invention permits amplification of nucleic acid molecules which are cotransferred to host cells with the nucleic acid encoding the replicase. Alternatively, the replicase may be encoded by the host cell. A preferred nucleic acid sequence encoding a replicase includes FHV RNA-1. RNA molecules can be prepared in vitro, e.g., using T7, T3 or SP6 polymerase, or by isolating RNA from virions or from cultured cells, e.g., rodent, Drosophilia or insect cells, that have been transfected or contacted with RNA or DNA molecules, or compositions, of the invention.
If systemic transfer of a gene of interest is desired, one of the nucleic acid sequences in a composition of the invention preferably encodes la CP, and optionally also encodes a MP, so that the vector is transmitted to the vascular tissue (the phloem). The expression of CP and MP can permit the synthesis of desired compounds in high yields, and may result in conferring disease resistance to all plant tissues. As described hereinbelow, a FHV-based vector for systemic infection of plants may be prepared which includes the 30 kDa movement protein of Tobacco Mosaic Virus (TMV MP; Deom et al., 1992) and the coat protein of a Red Clover Necrotic Mosaic Virus (RCNMV; Xiong et al., 1993). To monitor the movement of the vector through the plant, a marker gene may be introduced into the vector, e.g., a gene encoding green fluorescent protein (GFP, Epel et al., 1996; and Casper et al., 1996). GFP is a protein of 238 amino acid residues originally isolated from the jelly fish Aequorea victoria (Av). It absorbs blue light with maximal absorption at 395 nm and emits green light with peak emission at 590 nm. When GFP is expressed in either prokaryotic or eukaryotic cells, it is capable of producing a strong green fluorescence when excited by the blue light and this fluorescence requires no additional gene products from its host.
Thus, the vectors and compositions of the invention can be used to synthesize desired pharmacological compounds or other agents, e.g., agents encoding insect resistance, and for high throughput screening of uncharacterized genetic material, e.g., uncharacterized genetic material from a pesticide-resistant plant or insect nucleic acid encoding a useful trait. For example, uncharacterized genetic material from a pest-resistant plant is introduced into a vector of the invention, RNA is then expressed therefrom and a composition of the invention is prepared. Plants that are susceptible to the pest are exposed to the composition, preferably by spraying, and are then exposed to pest infestation. Leaves that exhibit signs of resistance are indicative that the introduced genetic material may encode pest resistance.
Thus, the invention also provides a quick assay system for developmental genes, regulatory genes, and genes for drug resistance and toxicity in insects and humans.
For applications in which a recombinant protein is to be expressed and then isolated from the host plant, e.g., a pharmaceutical compound, the composition of the invention is preferably applied to the plant by rubbing or spraying. For example, the contacted area of the plant may be such that only foliage of the plant is exposed to the composition, for example, plants such as tubers whose foliage has no commercial value. Once the protein is expressed in the plant, the recombinant protein(s) can be isolated and/or purified from the plant or its tissues.
Also provided is a method of expressing a preselected nucleic acid molecule in a host cell or host. The method comprises contacting a host cell or host with an amount of a composition of the invention. Preferred compositions include recombinant virus, isolated virion RNAs, in vitro-prepared RNA transcripts, or any combination thereof. Then the expression of the gene of interest is detected or determined. Hence, the invention further provides a transgenic host, the genome of which is augmented with the vector of the invention.
Preferred plant hosts for the gene transfer vectors and compositions of the invention include transgenic and non-transgenic monocots and dicots, e.g., Brassica, sweet corn, cucumber, barley, chenopodium, e.g., Chenopodium hybridum, cowpea, tobacco and Nicotiana benthamiana and the like. Preferably, the plant host is susceptible to Nodavirus infection.